Rhodamine-123 transport assay in organoids

The functional assay is a modified version of a previously described assay (54 (link)). Briefly, 1 week after seeding, the organoids were incubated with DMEM/F-12 (Invitrogen) containing 100 μM rhodamine-123 (Sigma) for 5 min, washed with three times with PBS, and supplemented with the regular medium. Images were taken every minute with a Leica SP-E confocal microscope for 30 min. Temperature and CO₂ concentration were kept at 37°C and 5%, respectively. To show that transport of rhodamine-123 depends on activity of multidrug-resistant (MDR) gene products, the organoids were incubated with 10 μM verapamil (Sigma), an MDR inhibitor, for 30 min before rhodamine-123 was added. As a negative control, gastric organoids were used, cultivated as previously described (28 (link)).